A SARS-CoV-2 virus strain (SARS-CoV-2/human/Korea/CNUHV03/2020) was propagated in Vero cells maintained at 37 °C in a humidified 5.0% CO${}_2$ incubator within a Biosafety Level-3 (BSL-3) facility. The facility (KCDC-08-3-03) was approved by the Korean government.

Korean ginseng (Panax ginseng Meyer) extract was manufactured by the Geumsan Ginseng & Herb Development Agency after purchasing a four-year-old root from Wooshin Industrial Co., Ltd. (Geumsan, Chungnam, Korea). The Korean ginseng was extracted with 70% ethyl alcohol twice at 70 °C for 6 h, concentrated in a vacuum at 70 °C to 16° Bx, and then spray-dried to obtain a powder. The Korean ginseng extract contained the following ginsenosides: Rg1 9.12 $\pm$ 0.08 mg/g, Re 15.91 $\pm$ 0.08 mg/g, Rf 4.57 $\pm$ 0.12 mg/g, Rb1 30.92 $\pm$ 0.08 mg/g, Rc 28.28 $\pm$ 0.55 mg/g, Rb2 19.23 $\pm$ 0.35 mg/g, and Rd 9.88 $\pm$ 0.26 mg/g.

Transgenic mice harboring human angiotensin-converting enzyme 2 (ACE2) [B6.Cg-Tg(K18-ACE2)2Prlmn/J] (ACE2-tg mouse) were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). Mice were bred in an animal facility at Chugnam National University, Korea.

Ten five-week-old female ACE2-tg mice were fed daily with a water containing ginseng extract (50 mg/kg body weight) for 180 days before being intranasally infected with 1 $\times$ 10${}^3$ plaque-forming units (PFU) of SARS-CoV-2/human/Korea/CNUHV03/2020. The infected mice were monitored for changes in body weight and mortality for 10 days.

ACE2-tg mice (n = 3 per group) were fed ginseng extract and infected with SARS-CoV-2 (1 $\times$ 10${}^3$ PFU), euthanized with a high dose of isoflurane USP (Gujarat, India) 6 days post-infection, and their lung tissues were collected. Lung tissue (0.1 g) was homogenized in 1 mL of phosphate-buffered saline (PBS) (pH 7.4) and centrifuged for 5 min at 12,300 g. RNA was isolated using the RNeasy Mini Kit (QIAGEN, Hilden, Germany).

The supernatant (100 $\mu$L) was then briefly disrupted in buffer RLT (350 $\mu$L) before adding 70% ethanol (550 $\mu$L). The sample (700 $\mu$L) was transferred to an RNeasy mini spin column and spun for 15 s at 12,300 g. The flow-through was discarded, and the RW1 buffer (700 $\mu$L) was added to the spin column prior to centrifugation for 15 s at 12,300 g. The flow-through was discarded, and the RPE buffer (500 $\mu$L) was added to the spin column prior to centrifugation for 15 s at 12,300 g. The spin column was placed in a new 1.5 mL collection tube before viral RNA was eluted using 40 $\mu$L of RNAse-free water.

To quantify the virus particles, we used the TaqMan real-time fluorescent PCR kit TOPrealTM One-step RT qPCR Kit (Enzynomics, Daejeon, Korea) and SARS-CoV-2 N primers and probe. The following components were mixed in a total volume of 20 $\mu$L:5 $\mu$L of the TOPrealTM One-step RT qPCR Kit (TaqMan probe), 1 $\mu$L of 10 pmol primers containing N_Sarbeco_Forward (5′-CACATTGGCACCCGCAAT-3′), N_Sarbeco-Reverse (5′-GAGGAACGAGAAGAGGCTTG-3′), N_Sarbeco_Probe (5′ FAM-ACTTCCTCAA GGAACAACATTGCCA-3′ BHQ1), 10 $\mu$L of viral RNA, and 2 $\mu$L of nuclease-free water. Real-time amplification was performed on the Rotor-Gene 6000 system (QIAGEN, Hilden, Germany) under the following reaction conditions: initial incubation at 50 °C for 30 min and 95 °C for 10 min, followed by 45 cycles of 95 °C for 5 sec and 60 °C for 30 sec. Viral titer (PFU) was calculated based on a standard curve generated using data for stock viruses with known PFU titers.

Lung tissues used for viral titration were fixed in 10% neutral-buffered formalin. The tissues were embedded in paraffin, and 0.5 $\mu$m thick tissue sections were prepared. Tissue sections were stained with hematoxylin and eosin (H&E) following the standard protocol. The stained tissue sections were observed under a DP70 light microscope (Olympus, Tokyo, Japan).

ACE2-tg mice (n = 3 per group) fed ginseng extract for 180 days were intranasally infected with SARS-CoV-2 (1 $\times$ 10${}^3$ PFU). Mice were euthanized with a high dose of isoflurane USP (Gujarat, India) six days post-infection before their lung tissues were collected. Lung tissue (0.1 g) was homogenized in PBS (1 mL, pH 7.4) and centrifuged for 5 min at 13,000 rpm.

The supernatants were obtained after centrifugation and used for the quantification of interferons, IFN-$\alpha$, IFN-$\beta$, and IFN-$\gamma$, using a Mouse IFN-$\alpha$ ELISA kit (Invitrogen, MA, USA), Veri${}^{\text{TM}}$Kine Mouse IFN-$\beta$ ELISA kit (Pestka), and Mouse IFN-$\gamma$ ELISA kit (Invitrogen, MA, USA), respectively. Assays were performed according to the manufacturer’s instructions. The microwell plates were washed twice with wash buffer. Assay buffer and diluted standards were added in duplicate to the wells of the plate. The sample diluent or supernatant of homogenized lung tissue was added in duplicate to the wells of the plate. The diluted biotin conjugate was added to all wells and incubated at room temperature (25 °C) for 2 h with shaking. The ELISA plates were washed four times with wash buffer. Diluted streptavidin-horseradish peroxidase was then added to each well. The plate was incubated at room temperature for 1 h on a microplate shaker, and the washing step was repeated as described above. TMB substrate was added to all the wells and incubated at room temperature for 30 min. The reaction was stopped by adding the stop solution to each microwell. The absorbance of each microwell was read using a microplate reader (Bio-Rad Laboratories, USA) at 450 nm as the primary wavelength. The amount of IFN was determined using a standard curve.

Statistical analysis was performed using the Student’s t-test with IBM SPSS Statistics version 20 (IBM Corp., Released 2011. Armonk, NY, USA). Statistical significance was set at p 0.05.

ACE2-tg mice (n = 10 per group) were fed ginseng extract (50 mg/kg) for 180 days and infected with 1 $\times$ 10${}^3$ PFU of SARS-CoV-2 virus. The infected mice were monitored for changes in body weight and mortality.

Ginseng-fed infected mice started to lose weight 6 days post-infection (p.i.), and infected mice that were not fed ginseng started to lose weight 4 days p.i. (Fig. 1A). The mean body weight percentage of ginseng-fed infected mice six days p.i. was 92.8% (p 0.05) of the original mean weight prior to infection, whereas that of the infected mice that were not fed ginseng was 79.4% of the original mean weight. Uninfected mice not fed ginseng were 103.2% of their original mean weight on day 6.

Fig. 1.

Body weight change and mortality in ACE2-tg mice that were fed ginseng and challenged with SARS-CoV-2. Female ACE2-tg mice (n = 10 per group) fed a diet containing ginseng extract (50 mg/kg body weight) for 180 days were intranasally infected with SARS-CoV-2 (1 $\times$ 10${}^3$ PFU). The infected mice were monitored for body weight change and mortality for 10 days. Statistical analysis was performed to compare ginseng-treated infected group and non-treated infected groups. (A) Body weight change in mice. (B) Mouse mortality. *p 0.05.

When mouse mortality was observed, all infected mice not fed ginseng died within 8 days p.i. Three out of the ten ginseng-fed infected mice survived, resulting in a 30% survival rate (p 0.05). All uninfected mice that were not fed ginseng survived (Fig. 1B).

ACE2-tg mice (n = 3 per group) were fed ginseng extract (50 mg/kg) for 180 days and infected with 1 $\times$ 10${}^3$ PFU of SARS-CoV-2 virus. The infected mice were euthanized on day 6 p.i. to measure viral titers (Fig. 2), and samples were harvested for pathological staining of lung tissues (Fig. 3).

Fig. 2.

Viral titers in lung tissues of ACE2-tg mice fed ginseng and challenged with SARS-CoV-2. ACE2-tg mice (n = 3 per group) fed a diet containing ginseng extract (50 mg/kg body weight) for 180 days were intranasally infected with SARS-CoV-2 (1 $\times$ 10${}^3$ PFU) and euthanized on day 6 post-infection prior to the collection of lung tissues. Lung tissues were homogenized in PBS (pH 7.4), and RNA was isolated with RNeasy Mini Kit. TaqMan real-time fluorescent PCR kit with specific primers of SARS-CoV-2 was used to quantify the virus particle by PFU. Viral titer (PFU) was calculated based on a standard curve generated using data for stock viruses with known PFU titers. Statistical analysis was performed to compare ginseng-treated infected group and non-treated infected groups. *p 0.05.

Fig. 3.

Lung pathology of ACE2-tg mice fed ginseng and challenged with SARS-CoV-2. The portions of lung tissues used for viral titration in Fig. 2 were fixed in neutral-buffered formalin and were embedded in paraffin. The tissues sections (0.5 $\mu$m thick) were stained with hematoxylin and eosin. The stained tissue sections were observed under a DP70 light microscope. (A) Lung tissue of uninfected mice not fed ginseng. (B) Lung tissue of infected mice not fed ginseng. (C) Lung tissue of ginseng-fed, infected mice.

The mean viral titer was much lower in the lung tissues of ginseng-fed infected mice than in those of mice that were not fed ginseng. The mean viral titer of ginseng-fed infected mice was 1.4 $\times$ 10${}^5$ PFU, while that of the infected mice that were not fed ginseng was 3.2 $\times$ 10${}^5$ PFU (Fig. 2).

The lung tissues of mice were stained with hematoxylin and eosin to determine the histopathological changes caused by SARS-CoV-2 infection (Fig. 3). The lung tissue of ginseng-fed infected mice showed much milder interstitial pneumonia (Fig. 3C) than that of infected mice not fed ginseng (Fig. 3B). The lung tissues of uninfected mice not fed ginseng did not show interstitial pneumonia (Fig. 3A).

Interferon content in the lung tissues of infected mice euthanized on day 6 p.i. was measured (Fig. 4) to explain the possible mechanism by which ginseng protects mice from SARS-CoV-2 infection. We measured interferons (IFN-$\alpha$, IFN-$\beta$, and IFN-$\gamma$), which have antiviral activity in the lungs of mice, using ELISA kits. Among the three interferons measured, IFN-$\beta$ (Fig. 4B) and IFN-$\gamma$ (Fig. 4C) were induced at much higher levels in the lung tissues of ginseng-fed infected mice than in infected mice that were not fed with ginseng. IFN-$\alpha$ was similarly induced in the lung tissues of ginseng-fed infected mice (0.1455 pg/0.1 g) and infected mice that were not fed ginseng (0.1255 pg/0.1 g) (Fig. 4A). The mean levels of IFN-$\beta$ in the lung tissues of ginseng-fed infected mice and infected mice that were not fed ginseng were 0.0162 pg/0.1 g and 0.011 pg/0.1 g, respectively (Fig. 4B). The mean IFN-$\gamma$ levels in the lung tissues of ginseng-fed infected mice and those not fed ginseng were 0.418 pg/0.1 g and 0.261 pg/0.1 g, respectively (Fig. 4C).

Fig. 4.

Interferon amount in lung tissues of ACE2-tg mice fed ginseng and challenged with SARS-CoV-2. ACE2-tg mice (n = 3 per group) were fed a diet containing ginseng extract (50 mg/kg body weight) for 180 days, intranasally infected with SARS-CoV-2 (1 $\times$ 10${}^3$ PFU), and euthanized on day 6 post-infection prior to the collection of lung tissues. Lung tissues were homogenized in PBS (pH 7.4) and centrifuged before supernatant collection. The amount of interferon in the supernatants of lung tissues was measured using mouse ELISA kits. Statistical analysis was performed to compare ginseng-treated infected group and non-treated infected groups. (A) IFN$\alpha$. (B) IFN$\beta$. (C) IFN$\gamma$. *p 0.05.